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Applied Microbiology and Biotechnology

, Volume 74, Issue 4, pp 791–804 | Cite as

Cloning and high-level production of a chitinase from Chromobacterium sp. and the role of conserved or nonconserved residues on its catalytic activity

  • Seur Kee Park
  • Chi Wook Kim
  • Hoon Kim
  • Jae Sung Jung
  • G. E. HarmanEmail author
Biotechnologically Relevant Enzymes and Proteins

Abstract

A gene encoding an alkaline (pI of 8.67) chitinase was cloned and sequenced from Chromobacterium sp. strain C-61. The gene was composed of 1,611 nucleotides and encoded a signal sequence of 26 N-terminal amino acids and a mature protein of 510 amino acids. Two chitinases of 54 and 52 kDa from both recombinant Escherichia coli and C-61 were detected on SDS-PAGE. Maximum chitinase activity was obtained in the culture supernatant of recombinant E. coli when cultivated in TB medium for 6 days at 37°C and was about fourfold higher than that from C-61. Chi54 from the culture supernatants could be purified by a single step based on isoelectric point. The purified Chi54 had about twofold higher binding affinity to chitin than to cellulose. The chi54 encoded a protein that included a type 3 chitin-binding domain belonging to group A and a family 18 catalytic domain belonging to subfamily A. In the catalytic domain, mutation of perfectly conserved residues and highly conserved residues resulted in loss of nearly all activity, while mutation of nonconserved residues resulted in enzymes that retained activity. In this process, a mutant (T218S) was obtained that had about 133% of the activity of the wild type, based on comparison of Kcat values.

Keywords

Chitinase Site-directed mutagenesis Enzyme purification Enzyme enhancement 

Notes

Acknowledgements

This study was supported by the Technology Development Program for Agricultural and Forestry, Ministry of Agriculture and Forestry, Republic of Korea. The authors thank Kristen Ondik and Michal Shoresh for editorial assistance.

References

  1. Blaak H, Schrempf H (1995) Binding and substrate specificities of a Streptomyces olivaceoviridis chitinase in comparison with its proteolytically processed form. Eur J Biochem 229:132–139PubMedGoogle Scholar
  2. Bokma E, Rozeboom HJ, Sibbald M, Dijkstra BW, Beintema JJ (2002) Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis. Eur J Biochem 269:893–901PubMedGoogle Scholar
  3. Bolar JP, Norelli JL, Harman GE, Brown SK, Aldwinckle HS (2001) Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants. Transgenic Res 10:533–543PubMedGoogle Scholar
  4. Broadway RM, Williams DL, Kain WC, Harman GE, Lorito M, Labeda DP (1995) Partial characterization of chitinolytic enzymes from Streptomyces albidoflavus. Lett Appl Microbiol 20:271–276PubMedGoogle Scholar
  5. Broadway R, Gongora C, Kain WC, Sanderson JP, Monroy JA, Bennett KC, Warner JB, Hoffman MP (1998) Novel chitinolytic enzymes with biological activity against herbivorous insects. J Chem Ecol 24:985–998Google Scholar
  6. Brun E, Moriaud F, Gans P, Blackledge MJ, Barras F, Marion D (1997) Solution structure of the cellulose-binding domain of the endoglucanase Z secreted by Erwinia chrysanthemi. Biochemistry 36:16074–16086PubMedGoogle Scholar
  7. Chernin L, Ismailov Z, Haran S, Chet I (1995) Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens. Appl Environ Microbiol 61:1720–1726PubMedPubMedCentralGoogle Scholar
  8. Ding X, Gopalakrishnan B, Johnson JB, White FF, Wang X, Morgan TD, Kramer KJ, Muthukrishnan S (1998) Insect resistance of transgenic tobacco expressing an insect chitinase gene. Transgenic Res 7:77–84PubMedGoogle Scholar
  9. Fang W, Leng B, Xiao Y, Jin K, Ma J, Fan Y, Feng J, Yang X, Zhang Y, Pei Y (2005) Cloning of Beauveria bassiana chitinase gene Bbchit1 and its application to improve fungal strain virulence. Appl Environ Microbiol 71:363–370PubMedPubMedCentralGoogle Scholar
  10. Flach J, Pilet PE, Jolles P (1992) What’s new in chitinase research? Experientia 48:701–716PubMedGoogle Scholar
  11. Gal SW, Choi JY, Kim CY, Cheong YH, Choi YJ, Lee SY, Bahk JD, Cho MJ (1998) Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme. FEMS Microbiol Lett 160:151–158PubMedGoogle Scholar
  12. Gilkes NR, Henrissat B, Kilburn DG, Miller RC Jr, Warren RA (1991) Domains in microbial β-1,4-glycanases; sequence conservation, function, and enzyme families. Microbiol Rev 55:303–315PubMedPubMedCentralGoogle Scholar
  13. Gleave AP, Taylor RK, Morris BA, Greenwood DR (1995) Cloning and sequencing of a gene encoding the 69-kDa extracellular chitinase of Janthinobacterium lividum. FEMS Microbiol Lett 131:279–288PubMedGoogle Scholar
  14. Hashimoto M, Ikegami T, Seino S, Ohuchi N, Fukada H, Sugiyama J, Shirakawa M, Watanabe T (2000) Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. J Bacteriol 182:3045–3054PubMedPubMedCentralGoogle Scholar
  15. Henrissat B (1999) Classification of chitinases modules. In: Jolles P, Muzzarelli RAA (eds) In Chitin and Chitinases. Birkhauser Verlag, Basel (1999), pp 137–156Google Scholar
  16. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7:637–644PubMedGoogle Scholar
  17. Ikegami T, Okada T, Hashimoto M, Seino S, Watanabe T, Shirakawa M (2000) Solution structure of the chitin-binding domain of Bacillus circulans WL-12 chitinase A1. J Biol Chem 275:13654–13661PubMedGoogle Scholar
  18. Jeuniaux C (1966) Chitinases. Methods Enzymol 8:644–650Google Scholar
  19. Kawase T, Saito A, Sato T, Kanai R, Fujii T, Nikaidou N, Miyashita K, Watanabe T (2004) Distribution and phylogenetic analysis of family 19 chitinases in Actinobacteria. Appl Environ Microbiol 70:1135–1144PubMedPubMedCentralGoogle Scholar
  20. Kobayashi DY, Reedy RM, Bick J, Oudemans PV (2002) Characterization of a chitinase from Stenotrophomonas maltophilia. Strain 34S1 and its involvement in biological control. Appl Environ Microbiol 68:1047–1054PubMedPubMedCentralGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685Google Scholar
  22. Lin FP, Chen HC, Lin CS (1999) Site-directed mutagenesis of Asp313, Glu315, and Asp391 residues in chitinase of Aeromonas caviae. IUBMB Life 48:199–204PubMedGoogle Scholar
  23. Lorito M, Harman GE, Hayes CK, Broadway RM, Tronsmo A, Woo SL, DiPietro A (1993) Chitinolytic enzymes produced by Trichoderma harzianum: antifungal activity of purified endochitinase and chitobiosadase. Phytopathology 83:302–307Google Scholar
  24. Lu Y, Zen KC, Muthukrishnan S, Kramer KJ (2002) Site-directed mutagenesis and functional analysis of active site acidic amino acid residues D142, D144 and E146 in Manduca sexta (tobacco hornworm) chitinase. Insect Biochem Mol Biol 32:1369–1382PubMedGoogle Scholar
  25. McGrew BR, Green DM (1990) Enhanced removal of detergent and recovery of enzymatic activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis: use of casein in gel wash buffer. Anal Biochem 189:68–74PubMedGoogle Scholar
  26. Morimoto K, Karita S, Kimura T, Sakka K, Ohmiya K (1997) Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain. J Bacteriol 179:7306–7314PubMedPubMedCentralGoogle Scholar
  27. Papanikolau Y, Prag G, Tavlas G, Vorgias CE, Oppenheim AB, Petratos K (2001) High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis. Biochemistry 40:11338–11343Google Scholar
  28. Park SK, Lee HY, Kim KC (1995a) Antagonistic effect of chitinolytic bacteria on soilborne plant pathogens. Korean J Plant Pathol 11:47–52Google Scholar
  29. Park SK, Lee HY, Huh JW (1995b) Production and some properties of chitinolytic enzymes by antagonistic bacteria. Korean J Plant Pathol 11:258–264Google Scholar
  30. Park SK, Lee HY, Kim KC (1995c) Role of chitinase produced by Chromobacterium violaceum in the suppression of Rhizoctonia damping-off. Korean J Plant Pathol 11:304–311Google Scholar
  31. Park SK, Lee MC, Harman ME (2005) The biocontrol activity of Chromobacterium sp. strain C-62 against Rhizoctonia solani depends on the productive ability of chitinase. Korean J Plant Pathol 21:275–282Google Scholar
  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  33. Shapiro M, Preisler HK, Robertson JL (1987) Enhancement of baculovirus activity of on gypsy moth (Lepidoptera; Lymantriidae) by chitinases. J Econ Entomol 80:1113–1116Google Scholar
  34. Simpson HD, Barras F (1999) Functional analysis of the carbohydrate-binding domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli putative chitinase. J Bacteriol 181:4611–4616PubMedPubMedCentralGoogle Scholar
  35. Simpson PJ, Xie H, Bolam DN, Gilbert HJ, Williamson MP (2000) The structural basis for the ligand specificity of family 2 carbohydrate-binding modules. J Biol Chem 275:41137–41142PubMedGoogle Scholar
  36. Suginta W, Vongsuwan A, Songsiriritthigul C, Svasti J, Prinz H (2005) Enzymatic properties of wild-type and active site mutants of chitinase A from Vibrio carchariae, as revealed by HPLC-MS. FEBS J 272:3376–3386PubMedGoogle Scholar
  37. Suzuki K, Taiyoji M, Sugawara N, Nikaidou N, Henrissat H, Watanabe T (1999) The third chitinase gene (chiC) of Serratia marcescens 2170 and the relationship of its product to other bacterial chitinases. Biochem J 343:587–596PubMedPubMedCentralGoogle Scholar
  38. Synstad B, Gaseidnes S, van Aalten DM, Vriend G, Nielsen JE, Eijsink VG (2004) Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase. Eur J Biochem 271:253–262PubMedPubMedCentralGoogle Scholar
  39. Tanaka T, Fujiwara S, Nishikori S, Fukui T, Takagi M, Imanaka T (1999) A unique chitinase with dual active sites and triple substrate binding sites from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1. Appl Environ Microbiol 65:5338–5344PubMedPubMedCentralGoogle Scholar
  40. Tanaka T, Fukui T, Imanaka T (2001) Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J Biol Chem 276:35629–35635PubMedGoogle Scholar
  41. Thomas CJ, Gooday GW, King LA, Possee RD (2000) Mutagenesis of the active site coding region of the Autographa californica nucleopolyhedrovirus chiA gene. J Gen Virol 81:1403–1411PubMedGoogle Scholar
  42. Tronsmo A, Harman GE (1993) Detection and quantification of N-acetyl-β-d-glucosaminidase, chitobiosidase and endochitinase in solutions and on gels. Anal Biochem 208:74–79PubMedGoogle Scholar
  43. Trudel J, Asselin A (1989) Detection of chitinase deacetylase activity after polyacrylamide gel electrophoresis. Anal Biochem 189:249–253Google Scholar
  44. Tsujibo H, Orikoshi H, Imada C, Okami Y, Miyamoto K, Inamori Y (1993) Site-directed mutagenesis of chitinase from Alteromonas sp. strain O-7. Biosci Biotechnol Biochem 57:1396–1407PubMedGoogle Scholar
  45. Tsujibo H, Orikoshi H, Shiotani K, Hayashi M, Umeda J, Miyamoto K, Imada C, Okami Y, Inamori Y (1998) Characterization of chitinase C from a marine bacterium, Altermonas sp. strain O-7, and its corresponding gene and domain structure. Appl Environ Microbiol 64:472–478PubMedPubMedCentralGoogle Scholar
  46. van Aalten DM, Synstad B, Brurberg MB, Hough E, Riise BW, Eijsink VG, Wierenga RK (2000) Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-Å resolution. Proc Natl Acad Sci U S A 97:5842–5847PubMedPubMedCentralGoogle Scholar
  47. Watanabe T, Kobori K, Miyashita K, Fujii T, Sakai H, Uchida M, Tanaka H (1993) Identification of glutamic acid 204 and aspartic 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J Biol Chem 268:18567–18572PubMedPubMedCentralGoogle Scholar
  48. Watanabe T, Ariga Y, Sato U, Toratani T, Hashimoto M, Nikaidou N, Kezuka Y, Nonaka T, Sugiyama J (2003) Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin. Biochem J 376:237–244PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Seur Kee Park
    • 1
  • Chi Wook Kim
    • 2
  • Hoon Kim
    • 3
  • Jae Sung Jung
    • 4
  • G. E. Harman
    • 5
    Email author
  1. 1.Department of Agricultural BiologySunchon National UniversitySunchonSouth Korea
  2. 2.Technical Research InstituteDong Bang Agro Corp.ChungNamSouth Korea
  3. 3.Department of Agricultural ChemistrySunchon National UniversitySunchonSouth Korea
  4. 4.Department of BiologySunchon National UniversitySunchonSouth Korea
  5. 5.Department of Horticultural SciencesCornell UniversityGenevaUSA

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